Process for the preparation of cyclic ketones

ABSTRACT

We describe a bioorganic process for the preparation of cyclopentanone and cyclopentenone derivatives of formula ##STR1## by β-oxidation of appropriate substrates, carried out by means of microorganisms. 
     The process is useful for preparation of 3-oxo-2-pentyl-1-cyclopentaneacetic and (Z)-3-oxo-2-(2-pentenyl)-1-cyclopentane-acetic acids and their methyl esters, compounds useful in the perfume and flavor industries.

TECHNICAL FIELD

The present invention relates to the field of biorganic synthesis. Itconcerns, more particularly, a process for the preparation of cyclicketones of formula ##STR2## optionally having a double bond in one ofthe positions indicated by the dotted lines and wherein: m represents aninteger from 0 to 3 and n an integer from 0 to 10; each of the symbolsR, which can be identical or different, stands for hydrogen or for asaturated or unsaturated, linear or branched, alkyl radical having 1 to6 carbon atoms; and each of the substituent groups can be located in anyavailable position of the ring; the process being characterized in thata substrate containing one or several cyclic carboxylic derivatives offormula ##STR3## wherein the dotted lines and the symbols R and m havethe meaning indicated in formula (I), p>n+2 and is defined as being aneven integer when n is even and an odd number when n is odd, is added toa culture of a microorganism capable of β-oxidising the fatty acid chainof said derivatives to form at least one of the desired ketones, whichis then extracted from the reaction medium.

PRIOR ART

Microbiological processes involving β-oxidation of substrates derivedfrom fatty acids are known in the prior art. For example, U.S. Pat. No.5,168,054 discloses a process of this type for the preparation oflactones starting from derivatives of linolenic, linoleic and oleicacids. However, to our knowledge, such processes have never been appliedto cyclic carboxylic derivatives such as those presently used.

DESCRIPTION OF THE INVENTION

We have now discovered that the process according to the presentinvention makes it possible to prepare a great variety of cyclopentenoneand cyclopentanone derivatives, in industrially applicable conditionsand very advantageous yields. The process of the invention is in fact ofvery wide application, both as regards the nature of the substrates andthe variety of microorganisms which can be used.

It has been established that one can use with equal success substrateswherein the fatty acid chain is located in the α or β position of thering, with respect to the ketonic group, and that said ring can furthercarry other substituent R groups, identical to or distinct from eachother.

Thus, according to a preferred and particularly advantageous embodimentof the invention, there is used a substrate of formula ##STR4## whereinthe dotted lines indicate the location of a single or double bond andp>n+2 and is an odd number. According to this preferred embodiment,there can thus be obtained compounds of formula ##STR5## n being an oddinteger, which compounds include cyclopentanone derivatives useful forthe synthesis of fragrant molecules. For example,3-oxo-2-(2-pentenyl)-1-cyclopentaneacetic acid, or jasmonic acid, is aprecursor of methyl jasmonate, a compound much appreciated in perfumeryand a natural component of jasmine essential oil.

Likewise, 3-oxo-2-pentyl-1-cyclopentaneacetic acid, or dihydrojasmonicacid, is a precursor of Hedione® (methyl3-oxo-2-pentyl-1-cyclopentaneacetate; origin: Firmenich SA, Geneva,Switzerland), a perfuming ingredient much valued for its floral,jasmine-like fragrance and its exhalting odor effect.

In addition to their use fullness in perfumery, these acids and theirmethyl esters are also useful for the flavor industry, in thepreparation of edible products, and there is therefore a real need for abioorganic process allowing their preparation via a microbiologicalpathway.

Furthermore, it is also known that several varieties of plants arecapable of producing these compounds and that jasmonic acid and methyljasmonate in particular play an important role in the metabolism of suchplants, namely as growth regulators. This has prompted the interest ofseveral investigators to study their biosynthesis, occurring via theaction of enzymes present in the corresponding vegetable tissues (seefor example, B. A. Vick et al., Plant Physiol. 1984, 75, 458-61).However, it is clear that the isolation of the above-mentioned compoundsfrom such vegetable tissues is not an economically viable synthesis on alarge scale.

Now, the process according to the present invention brings precisely anovel solution to the problem of the bioorganic synthesis of thesecompounds. Thanks to the instant process, the compounds of formulae (I)and (Ia) can be prepared in excellent yields, by means of a largevariety of microorganisms, which organisms were unknown heretofore fortheir capability to metabolise the formula (II) substrates, and moreparticularly the formula (IIa) substrates which make it possible toobtain jasmonic and dihydrojasmonic acids. Therefore, it could not havebeen predicted that a process such as presently claimed could open a newand general pathway for the preparation of cyclopentenone andcyclopentanone derivatives, which turns out to be particularly usefulfor the synthesis of ingredients of great value in the fragrance andfood industries.

In addition, it has been observed that particular embodiments of theprocess of the invention can favor the formation of specific isomers ofcompounds (Ia). In fact, as a result of their structure, these compoundscan assume two stereoisomeric forms of cyclanic cis or transconfiguration, each of which possesses two enantiomers.

It should however be noted that, although the production of jasmonic anddihydrojasmonic acids is one of the main aims of the invention, it isnevertheless the case that the process presently disclosed is of a farmore general application and allows the preparation of a large varietyof cyclopentenone and cyclopentanone derivatives.

For example, according to another embodiment of the invention, there isused as substrate a compound of formula ##STR6## wherein p has themeaning indicated in formula (II), to obtain 2-oxo-1-cyclopentaneaceticacid and higher homologues thereof.

In actual fact, there seems to be no limitation to the nature of thesubstrates which can be transformed according to the processabove-described. Thus, one can also use substrates possessing evenlonger fatty acid side chains than those cited above, and such chainsmay optionally be unsaturated and even carry lower alkyl radicals,namely methyl and ethyl radicals, as substituents. It should be notedthat, when the substrate possesses a very long fatty acid chain, theprocess of the invention makes it possible to convert it into severallower homologues, which are formed in sequential reactions and which canthen themselves be converted into lower homologues, until formation ofthe corresponding compounds (I) having n=0 or 1. In this manner, theoxidation product can be formed of one or several such metabolites fromthe reaction sequence, depending on the reaction time and on the kineticcharacteristics of the successive β-oxidations. If desired, once thereaction mixture has been extracted from the medium, the variouscomponents of this mixture can be separated via the usual methods, suchas chromatography or distillation. Alternatively, and according to thenature of the product that is desired to obtain, the microorganism isallowed to act until all the intermediate metabolites have beenconverted at once, to collect essentially only the last product of thereaction sequence.

In this way, one can for instance obtain essentially jasmonic acid, or2-(2-pentenyl)-3-oxo-1-cyclopentaneacetic acid, starting from3-oxo-2-[2-pentenyl]-1-cyclopentaneoctanoic acid, a natural component ofcertain plants, or yet starting from one of its lower homologues. Suchconversions are described in detail in the examples presented furtheron.

Amongst the microorganisms that can be used according to the inventionto carry out the β-oxidation of substrates (II), those of theSaccharomyces or Rhodococcus genus turned out to be particularlyadvantageous, namely for the preparation of jasmonic and dihydrojasmonicacids.

As preferred microorganisms to be used according to the invention, therecan be further cited those selected from the group consisting ofRhodococcus rhodochorus, Rhodococcus erytropolis, Rhodococcus sp.,Nocardia calcarea, Arthrobacter petroleophagus, Arthrobacterartrocyanus, Arthrobacter ureafaciens, Aspergillus niger, Saccharomycescerivisae, Mycobacteriurn phlei, Streptomyces viridosporus, Streptomycesrosechromogenus, Streptomyces bacilliaris, Cylindrocarpon candidum,Escherichia coli, Hansenula polyrmorpha, Pseudomonas Sp., Serratiamarcesens et Aspergillus oryzae. Such microorganisms can be obtainedfrom Internationally recognized deposit authorities. In the particularcase of Saccharomyces, they can be bought from specialized firms, whichfrequently provide locally cultured strains, stemming from the beer,wine or baking industries. All these species are quite convenient forthe process according to the invention. Cultures of these microorganismsare obtained in the conventional manner. Their prior growth is carriedout in current nutritive mediae and under the usual conditions, eitherin stationary phase or under stirring.

The cells are then isolated from the culture medium, for example bycentrifuging, and suspended in an aqueous medium containing theabove-mentioned substrates and generally devoid of any other nutritiveor metabolisable source, at temperatures preferably comprised between20° and 35° C., for variable but relatively short periods of times,typically comprised between 24 and 72 hours, under aerobic conditions.During the reaction, the pH of the medium will be preferably maintainedbetween 5 and 10.

According to a particular embodiment of the invention, before adding thesubstrate to the microorganism culture, there is provided a first stepof preparation via aeration of the culture for a period of time, whichstep consists in suspending said culture in water, under aerobicconditions and stirring, at a temperature comprised between 15° and 30°C., for an amount of time sufficient to ensure that any nutritivesource, of endogenous or exogenous origin, has been entirely consumedbefore addition of the substrates of formula (II). Such a preparationstep is particularly appropriate whenever the microorganism culture, asa result of its growth conditions, possesses a residual nutritivesource, namely a carbohydrate source, as is typically the case ofSaccharomyces for example.

The substrate is then added to the microorganism culture thus prepared,such addition being preferably carried out in the absence of any othernutritive source, under the conditions cited above.

The cells are then separated from the reaction medium by centrifugationor ultrafiltration and the aqueous solution thus obtained acidified andrepeatedly extracted by means of an appropriate solvent, e.g. diethylether. The combined organic layers are then treated in the usual mannerand, if desired, their components (I) separated by chromatography.

It should be noted that the acids thus obtained can then be readilyesterified by chemical or enzymatic means to form the correspondingesters.

The substrates of formula (II) are either commercial origin compounds orcan be easily prepared from commercial products. For example, thesubstrates derived from cyclopentanone can be conventionally preparedvia addition reactions on cyclopent-2-en-1-one, or derivatives thereof,according to the following general scheme: ##STR7## the fatty acid chainbeing introduced in the form of a group acting as an electrophile or asa nucleophile, depending on the ring position into which one wishes tointroduce it and on the conditions of the addition reaction. Forexample, the formula (IIa) substrates having a saturated ring can beprepared following the scheme hereinafter: ##STR8## In this scheme, thedotted line and p have the meaning indicated in formula (IIa). Thestarting cyclopentenone is a known compound (see, for example, EP 110142) and the brominated reagent can be obtained in conventional mannerand as described further on, starting from commercial origin diols.

As regards the formula (II) substrates which possess an endocylic doublebond, they can be obtained in analogous manner to that described forexample by P. A. Grieco et al. in J. Org. Chem. 1989, 54, 6008-6010, andas represented hereinafter: ##STR9##

The conditions of these conventional reactions are described in detailin the examples presented further on.

Clearly, the substrates of formula (II) can be used in the form ofracemic mixtures of cis and trans configuration stereoisomers, as cisand trans configuration racemates, or yet in the form of any one of thefour possible diastereomers in pure state. It was observed that certainmicrorganisms, when put into contact with racemic substrates, containingmixtures of cis and trans isomers, favored the formation of particularchiral final products. The details of such transformations are presentedin the following examples.

The process of the invention will now be described in further detail byway of the following examples, wherein the temperatures are indicated indegrees centigrade and the abbreviations have the ususal meaning in theart. In the Tables, the cited amounts of the products obtained areindicated in percentage, relative to an internal standard and asmeasured by chromatographic analysis.

EMBODIMENTS OF THE INVENTION EXAMPLE 1 General Method I

A 5 neck flask (200 ml), fitted with a Medimix® type turbine, a pHmeasuring cell, an air inlet, a condenser and a sampling outlet, wascharged with the microorganism culture (10 g of wet biomass) and 50 mlof demineralised water. The cell suspension was aerated (1 v/v/m) andstirred (1,000 rpm) overnight at 30°. The suspension pH was thenadjusted to 5.5 and a solution of the substrate to be used, in thepresent case 3-oxo-2-pentyl-1-cyclopentaneoctanoic acid (100 mg, 0.34mmole, 2 g/l; see preparation further on), in water (10 ml) was addedthereto. The reaction was followed by taking aliquots (1 ml) of themixture at regular intervals. Such samples were centrifuged in plastictubes of the Eppendorf type during 2 minutes to precipitate the cells.An aliquot (0.45 ml) of the supernatant transparent solution was thenacidified with HCl 5M (0.05-0.1 ml) and extracted with diethyl ether (2times, identical volumes). The organic extracts were combined, driedunder nitrogen and treated with diazomethane to form the methyl estersof the acids contained in the reaction product. This mixture of esterswas then analysed by gas chromatography. After a 24 h reaction, thereaction mixture was centrifuged (20 min, at 10,000 rpm) to precipitatethe cells and the supernatant liquid was decanted. In order to wash thecells, the latter were once again suspended in 50 ml of water andstirred for 30 min. After recentrifuging, there was obtained a secondsupernatant liquid which was combined with the first and the pH of whichwas adjusted to 12 with NaOH (10%). The resulting solution was extractedwith diethyl ether (3 times, identical volumes) to separate the neutralfraction. The pH was then adjusted to 2 with HCl (5M) and the acidicfraction collected by extraction. The combined organic extracts weredried over Na₂ SO₄ and the solvant stripped under vacuum. An aliquot wasthen methylated by means of diazomethane and the product thus obtainedanalysed by gas chromatography [SPB5 type column, 30 m length, 0.32 mminternal diameter, 50°(0')-230°(5') at 10°/min; sample dissolved in 0.1ml of diisopropylether containing 1 g/l of methyl myristate as internalstandard--retention time 13.91 minutes].

Upon a test carried out on a scale 5 times larger, using Saccharomycescerevisiae (origin: Here, Schweiz, AG, 9507 Stettfurt, Switzerland), thefollowing compounds were isolated and identified [GC-MS: M/Z (% relativeto the internal standard)]:

1. methyl 3-oxo-2-pentyl-1-cyclopentaneacetate

trans isomer: retention time--13.18 min

GC-MS: 226 (3) [M⁺ ], 195 (2) [M--OCH₃ ⁺ ], 156 (35) [M--C₅ H₁₁ +H⁺ ],153 (31) [M--CH₂ --COOCH₃ ⁺ ], 109 (5), 96 (10), 83 (100) [C₅ H₆ O+H⁺ ],82 (24) [C₅ H₆ O⁺ ], 55(13)

cis isomer: retention time--13.46 min

GC-MS: 226 (5) [M⁺ ], 156 (25) [M--C₅ H₁₁ +H⁺ ], 153 (29) [M--CH₂ COOCH₃⁺ ], 109 (5), 103 (15), 96 (11), 83 (100) [C₅ H₆ O+H⁺ ], 82 (24) [C₅ H₆O⁺ ], 55 (19)

2. methyl 3-oxo-2-pentyl-1-cyclopentanebutanoate

trans isomer: retention time--15.53 min

GC-MS: 254 (1) [M⁺ ], 184 (13) [M--C₅ H₁₁ +H⁺ ], 153 (29) [M--(CH₂)₃COOCH₃ ⁺ ], 109 (4), 97 (5), 83 (100) [C₅ H₆ O+H⁺ ], 82 (30) [C₅ H₆ O⁺], 55 (10)

cis isomer: retention time--15.72 min

GC-MS: 254 (1) [M⁺ ], 184 (13) [M--C₅ H₁₁ +H⁺ ], 153 (29) [M--(CH₂)₃COOCH₃ ⁺ ], 109 (4), 97 (5), 83 (100) [C₅ H₆ O+H⁺ ], 82 (29) [C₅ H₆ O⁺], 55 (10)

3. methyl 3-oxo-2-pentyl-1-cyclopentanehexanoate

trans isomer: retention time--17.42 min

GC-MS: 282 (1) [M⁺ ], 251 (4) [M--OCH₃ ⁺ ], 212 (13) [M--C₅ H₁₁ +H⁺ ],153 (18) [M--(CH₂)₅ COOCH₃ ⁺ ], 130 (29), 83 (100) [C₅ H₆ O+H⁺ ], 82 (8)[C₅ H₆ O⁺ ], 55 (11)

cis isomer: retention time--17.65 min

4. methyl 3-oxo-2-pentyl-1-cyclopentaneoctanoate

trans isomer: retention time--19.35 min

GC-MS: 310 (1) [M⁺ ], 279 (2) [M--OCH₃ ⁺ ], 240 (11) [M--C₅ H₁₁ +H⁺ ],153 (31) [M--(CH₂)₇ COOCH₃ ⁺ ], 83 (100) [C₅ H₆ O+H⁺ ], 82 (38) [C₅ H₆O⁺ ], 55 (10)

Preparation of the Starting Product

The starting product in the above-described reaction, i.e.3-oxo-2-pentyl-1-cyclopentaneoctanoic acid, was prepared as follows. Asolution of 1,8-octanediol (15 g, 0.1 mole) in toluene (235 ml) and THF(tetrahydrofuran, 15 ml) was added dropwise to a NaH slurry (oildispersion 65-70%, 5.3 g, 0.15 mole) in toluene (50 ml), kept understirring and N₂, at room temperature. The mixture was heated to refluxduring 60 h and benzyl chloride (33 g, 0.26 mole) was then added and themixture kept at reflux for yet 60 h. The cooled reaction mixture waspoured on cold sat. aq. NH₄ Cl and extracted with ether. The combinedorganic phases were washed with aq. sat. NaCl, dried over anhydrous Na₂SO₄ and concentrated under vacuum to provide a pale yellow oil (24.5 g).After chromatography on silica column (350 g), with cyclohexane/ethylacetate 4:1 as eluting agent, followed by bulb-to-bulb distillationunder vacuum, pure 8-benzyloxyoctan-1-ol was obtained, in the form of apale yellow oil (11.9 g, yield 49%).

B.p.: 200°-220° (bath)/4 Pa Rf (cyclohexane/ethyl acetate 7:3) 0.36 IR(CDCl₃): 3450 (broad), 3010, 2934, 2858, 1454, 1095 cm⁻¹ NMR (¹ H, 360MHz, D₂ O): 1.25-1.45(8H); 1.45-1.70(4H); 3.46(t, J=7 Hz, 2H); 3.60(t,J=7 Hz, 2H); 4.50(s, 2H); 7.23-7.38(5H) δ ppm NMR (¹³ C): 138.6(s);128.3(d); 127.6(d); 127.5(d); 72.9(t); 70.5(t); 62.9(t); 32.7(t);29.7(t); 29.4(2t); 28.1(t); 25.7(t) δ ppm MS: 236 (2, M⁺), 107(59),91(100)

To a stirred solution of this compound (11.6 g, 0.049 mole) and pyridine(9.8 g, 0.12 mole) in CH₂ Cl₂ (100 ml), there was added by portions, atroom temperature (r.t.) and under N₂, tosyl chloride (12.8 g, 0.067mole). After 20 h at r.t., the mixture was poured on 5% aq. HCl, cooledand extracted (CH₂ Cl₂). The organic phase was washed with sat. aq.NaHCO₃ and brine, dried over anhydrous Na₂ SO₄ and concentrated. Thesemi-crystalline residual oil (19.2 g; raw tosylate of theabove-mentioned benzyloxyoctanol) was retaken in acetone (400 ml) andLiBr (10.9 g, 0.126 mole) added thereto. The stirred mixture was thentaken to reflux during 90 min, cooled to r.t. and filtered. The filtratewas concentrated and dissolved in ether (150 ml). This organic phase wasthen successively washed with H₂ O and aq. sat. NaCl and dried overanhydrous Na₂ SO₄. Concentration under vacuum (80°/8 Pa) provided1-benzyloxy-8-bromooctane in the form of a pale yellow oil (12.4 g;yield 84%).

IR (CHCl₃): 3011, 2934, 2858, 1454, 1364, 1098 cm⁻¹ NMR (¹ H, 360 MHz):1.25-1.50(8H); 1.60(m, 2H); 1.83(m, 2H); 3.39(t, J=7 Hz, 2H); 3.46(t,J=7 Hz, 2H); 4.50(s, 2H); 7.22-7.37(5H) δ ppm NMR (¹³ C): 138.7(s);128.3(d); 127.6(d); 127.5(d); 72.9(t); 70.4(t); 33.9(t); 32.8(t);29.7(t); 29.3(t); 28.7(t); 28.1(t); 26.1(t) δ ppm MS: 298 (11, M⁺),207(52), 188(17), 147(16), 109(22), 9(100)

A solution of this bromooctane (11 g, 0.037 mole) in ether (36 ml) wasadded dropwise to a stirred slurry of Mg turnings (0.91 g, 0.037 mole)in ether (4 ml), at r.t. and under N₂. The resulting Grignard reagentwas heated to reflux for 30 min, then cooled and added dropwise to aslurry of CuBr.(CH₃)₂ S (3.2 g, 0.015 mole) in ether (30 ml), understirring and at -44°. After 3 min, a solution of2-pentylcyclopent-2-en-1-one (4.7 g, 0.028 mole; see EP 110 142, forexample) in ether (15 ml) was added dropwise during 30 min, at -44°.After an additional 15 min at -40°, the mixture was allowed to attain 0°during 1 h and then poured over cold aq. NH₄ Cl. Extraction with etherafforded an organic phase which was successively washed with H₂ O andsat. aq. NaCl and dried over anhydrous Na₂ SO₄. Concentration undervacuum afforded a semi-crystalline green oil (12.7 g) which was purifiedby chromatography [SiO₂ (280 g) ; eluting agent: cyclohexane/ethylacetate 19:1)] to afford3-(8'-benzyloxyoct-1'-yl)-2-pentylcyclopentan-1-one (12:1 trans/cismixture) in the form of a pale yellow oil (3.1 g; yield 30%), which wasdried at 100°/7.9 Pa.

R_(f) (cyclohexane/ethyl acetate 9:1) 0.43 IR (CHCl₃): 3011, 2930, 2857,1732, 1455, 1098 cm⁻¹ NMR (¹ H, 360 MHz): 0.88(t, J=7 Hz, 3H);1.20-2.40(28H); 3.47(t, J=7 Hz, 2H); 4.50(s, 2H); 7.23-7.37(5H) δ ppmNMR (¹³ C): 221.5(s); 138.7(s); 128.3(d); 127.6(d); 127.5(d); 72.9(t);70.5(t); 55.1(d); 41.6(d); 37.9(t); 34.8(t); 32.2(t); 29.8(t); 29.6(t);29.5(t); 28.1(t); 27.1(t); 26.6(t); 26.2(t); 22.5(t); 14.1(q) δ ppm MS:372 (6, M⁺), 243(6), 196(8), 173(14), 153(35), 91(100), 83(92)

A solution of this cyclopentanone (3 g, 8.05 mmole; trans/cis 12:1) inethanol (30 ml) containing 10% Pd--C (0.24 g), was hydrogenolysed atr.t. during 4 h. After filtration (Hyflo®), concentration under vacuumand bulb-to-bulb distillation,3-(8'-hydroxy-1'-octyl)-2-pentylcyclopentan-1-one was obtained (12:1trans/cis mixture) in the form of a pale yellow oil (2.1 g; yield 92%).

B.p.: 230°-240° (bath)/5.3 Pa IR (CHCl₃): 3437 (broad), 2930, 2857,1732, 1456, 1160, 1051 cm⁻¹ NMR (¹ H, 360 MHz, D₂ O): 0.88(t, 3H);1.20-2.00(24H); 2.00-2.36(4H); 3.65(t, J=7 Hz, 2H) δ ppm NMR (¹³ C):221.6(s); 63.0(t); 53.1(d); 41.6(d); 37.9(t); 34.8(t); 32.8(t); 32.2(t);29.8(t); 29.6(t); 29.4(t); 28.1(t); 27.1(2t); 26.6(t); 25.8(t); 22.5(t);14.1(q) δ ppm MS: 282 (0, M⁺), 212(4), 153(17), 83(100)

Jones reagent (H₂ CrO₄ /aq. H₂ SO₄ ; 7.2 ml of a 2.5M solution) wasadded dropwise to a stirred solution of the latter cyclopentanone (2 g,7.1 mmole; trans/cis 12:1) in acetone (40 ml) at 20°, under N₂. After afurther 40 min at 22°-24°, the mixture was poured into water andextracted with ether. The organic phase was successively washed with H₂O, 10% aq. NaCl and sat. aq. NH₄ Cl, dried over anhydrous Na₂ SO₄ andconcentrated under vacuum. The resulting yellow oil (2.1 g) was purifiedby chromatogarphy [SiO₂ (100 g); eluting agent:cyclohexane/ethyl acetate1:1.5)] to afford the desired 3-oxo-2-pentyl-1-cyclopentaneoctanoic acid(trans/cis 12:1) as a viscous pale yellow oil (1.9 g; yield 90%).

IR (CHCl₃): 3050 (broad), 2931, 2858, 1732, 1710, 1460, 1160 cm⁻¹ NMR (¹H, 360 MHz, D₂ O): 0.89(t, J=7 Hz, 3H); 1.20-2.35(28H); 2.37(t, J=7 Hz,2H) δ ppm NMR (¹³ C): 221.8(s); 179.6(s); 55.1(d); 41.6(d); 37.9(t);34.8(t); 34.0(t); 32.2(t); 29.6(t); 29.2(t); 29.0(t); 28.1(2t); 27.1(t);26.6(t); 24.7(t); 22.5(t); 14.1(q) δ ppm MS: 297 (2, M⁺), 279(3),226(9), 153(17), 83(100)

EXAMPLE 2

We proceeded according to the general method I described in Example 1,using a variety of different microorganisms, cited in the table below.In this table, the compounds are indicated by the number attributed tothem in Example 1. These results relate to the product obtained after 24h of reaction, i.e. of activity of the corresponding microorganism. Theyield in compound 1 represents the molar % relative to compound 4, basedon the amount of the corresponding starting acid (100 mg).

                  TABLE I                                                         ______________________________________                                                 Weight of              Yield in                                               acid extract                                                                          Compound (% GC)                                                                              compound 1                                    Microorganism                                                                            (mg)      1      2    3   4    (molar %)                           ______________________________________                                        Serratia marcesens                                                                       36.6      84.5   11.9 1.4  2.2 43.2                                ATCC 8100                                                                     Aspergillus niger                                                                        25.0      74.4    9.8 6.0  9.8 26.0                                ATCC 16888                                                                    Escherichia coli                                                                         35.8      49.0   23.2 4.8 23.1 35.8                                ATCC 8677                                                                     Saccharomyces                                                                            35.7      31.8   54.0 9.1  5.0 15.9                                cerevisiae                                                                    Streptomyces                                                                             34.3      30.4   25.9 8.4 35.3 14.6                                viriosporus                                                                   ATCC 39115                                                                    Hansenula  --        13.0   52.7 8.7 25.5 --                                  polymorpha                                                                    Pseudomonas Sp.                                                                          --        0.0     2.4 2.5 95.1 --                                  DMS 1650                                                                      ______________________________________                                    

This table shows that all the microorganisms were able to carry out theβ-oxidation of the 3-oxo-2-pentyl-1-cyclopentaneoctanoic, -hexanoic and-butanoic acids. The yields in final acid, i.e.3-oxo-2-pentyl-1-cyclopentaneacetic acid, and thus in the correspondingcompound 1, can of course be distinctly improved by prolonging thereaction beyond 24 h. Furthermore, the described extraction proceduremay also be improved to minimize the losses in the desired products.

EXAMPLE 3 General Method II

The microorganism to be used was prior grown on a starch-containingmedium for three days. At the end of the three days, all the starch hadbeen consumed. The cells were collected by centrifugation and a portionof the resulting biomass (2 g), as well as 8 ml of demineralized water,were placed in an Erlenmeyer type flask (50 ml) containing the substrateto be used, in the present case(Z)-3-oxo-2-(2-pentenyl)-1-cyclopentaneoctanoic acid (20 mg, 2 g/l; seepreparation further on). The vial was sealed with a cotton wool bung andplaced on an orbital shaker for 24 h, at 30° and 140 rpm. The cells werethen spun down by centrifugation (10,000 rpm) for 15 min at r.t. Afterremoving the supernatant with a pipette, the cells were resuspended indemineralized water (10 ml) and then spun down again as above. Theresulting supernatant was combined with that first obtained asabove-described and the combined volume adjusted to 25 ml withde-ionized water. This solution was then brought to pH 2 with H₂ SO₄(10%) and extracted with diethyl ether (1×50 ml et 1×25 ml). Thecombined organic phases were dried over anhydrous MgSO₄. The solvent wasstripped under nitrogen flow and the product weighed. It was thentreated with diazomethane to afford the corresponding methyl esters. Theproduct thus obtained was anlyzed by chromatography, relative to anexternal standard. To this end, a solution in 1 ml of methylpentadecanoate (5 g/l) was prepared and chromatographed [SPB5 column, 30m length, 0.32 mm internal diameter, 80°(0')-230°(20') at 15°/min]. Theamount of each of the components was assessed on the basis of the areaof the corresponding peak, relative to that of the standard. Proceedingas described above, upon a reaction carried out with Saccharomycescerevisiae (origin: Switzerland), the following products were obtainedand identified.

A. methyl (Z)-3-oxo-2-(2-pentenyl)-1-cyclopentaneacetate

trans isomer: retention time--10.28 min

GC-MS: 224 (31) [M⁺ ], 206 (4) [M--H₂ O⁺ ], 193 (10) [M--OCH₃ ⁺ ], 177(6), 156 (23) [M--C₅ H₉ +H⁺ ], 151 (31) [M--CH₂ COOCH₃ +], 109 (24), 95(30),83 (100)[C₅ H₆ O+H⁺ ], 55 (37), 41 (73)

cis isomer: retention time--10.56 min

GC-MS: 224 (24) [M⁺ ], 206 (12) [M--H₂ O⁺ ], 177 (13), 156 (15) [M--C₅H₉ +H⁺ ], 151 (31) [M--CH₂ COOCH₃ ⁺ ], 109(23), 95 (42), 83 (100) [C₅ H₆O+H⁺ ], 55 (19), 41 (81)

B. methyl (Z)-3-oxo-2-(2-pentenyl)-1-cyclopentanebutanoate

trans isomer: retention time--12.74 min

GC-MS: 252 (17) [M⁺ ], 234 (7) [M--H₂ O⁺ ], 184 (10) [M--C₅ H₉ +H⁺ ],151 (59) [M--(CH₂)₃ COOH₃ ⁺ ], 109 (22), 95 (35), 83 (100) [C₅ H₆ O+H⁺], 41 (67)

cis isomer: retention time--13.10 min

GC-MS: 252 (8) [M⁺ ], 234 (20) [M--H₂ O⁺ ], 205 (10), 184 (9) [M--C₅ H₉+H⁺ ], 151 (37) [M--(CH₂)₃ COOH₃ ⁺ ], 109 (30), 95 (47), 83 (100) [C₅ H₆O+H⁺ ], 41 (73)

C. methyl (Z)-3-oxo-2-(2-pentenyl)-1-cyclopentanehexanoate

trans isomer: retention time--16.37 min

GC-MS: 280 (7) [M⁺ ], 262 (8) [M--H₂ O+], 212 (16) [M--C₅ H₉ +H⁺ ], 151(39) [M--(CH₂)₅ COOCH₃ ⁺ ], 95 (40), 83 (100) [C₅ H₆ O+H⁺ ], 82 (8) [C₅H₆ O⁺ ], 41 (55)

cis isomer: retention time--17.10 min

D. methyl (Z)-3-oxo-2-(2-pentenyl)-1-cyclopentaneoctanoate

trans isomer: retention time--22.71 min

GC-MS: 308 (4) [M⁺ ], 290 (4) [M--H₂ O⁺ ], 277 (7) [M--OCH₃ ⁺ ], 240(18) [M--C₅ H₉ +H⁺ ], 151 (43) [M--(CH₂)₇ COOCH₃ ⁺ ], 124 (35), 95 (48),83 (100) [C₅ H₆ O+H⁺ ], 55 (43)

cis isomer: retention time--23.82 min

GC-MS: 308 (3) [M⁺ ], 290 (4) [M--H₂ O⁺ ], 277 (5) [M--OCH₃ ⁺ ], 240(14) [M--C₅ H₉ +H⁺ ], 151 (37) [M--(CH₂)₇ COOCH₃ ⁺ ], 124 (40), 95 (50),83 (100) [C₅ H₆ O+H⁺ ], 55 (24)

The reaction product was also analyzed on a chiral column of theMegadex® dimethylpentyl-β-cyclodextrine type (10 m×2.5 mm×0.25 μm filmthickness) using a temperature program of 50°(1')-130°(15'), heating15°/min, and up to 220°(5'), heating 2°/min. The retention times of eachof the chiral species corresponding to each of compounds A, B, C and Dabove are indicated in the following table.

                  TABLE II                                                        ______________________________________                                                       Retention time                                                 Chiral isomer  (min)                                                          ______________________________________                                        (-)-trans-A    17.61                                                          (+)-trans-A    18.93                                                          (+)-cis-A      20.29                                                          (-)-cis-A      20.29                                                          (-)-trans-B    33.78                                                          (+)-trans-B    34.83                                                          (+)-cis-B      35.86                                                          (-)-cis-B      35.86                                                          (-)-trans-C    44.57                                                          (+)-trans-C    45.16                                                          (+)-cis-C      46.32                                                          (-)-cis-C      46.32                                                          (-)-trans-D    53.7-54.1                                                      (+)-trans-D    53.7-54.1                                                      (+)-cis-D      55.21                                                          (-)-cis-D      55.21                                                          ______________________________________                                    

Preparation of the Starting Product

The starting product in the reaction described above, i.e.(Z)-3-oxo-2-(2-pentenyl)-1-cyclopentaneoctanoic acid, was prepared asfollows. A solution of 1-benzyloxy-8-bromooctane (5 g, 0.015 mole; seeExample 1) in ether (15 ml) was added dropwise to a stirred suspensionof Mg turnings (0.38 g, 0.016 mole) in ether (2 ml), at r.t. under N₂.The resulting Grignard reagent was heated to reflux for a further 30min, then cooled and added dropwise to a slurry of CuBr.(CH₃)₂ S (1.25g, 6 mmole) and LiBr (1.9 g, 0.022 mole) in THF (15 ml), under stirringand at -44°. After 3 min, a solution of(Z)-2-(2-pentenyl)-cyclopent-2-en-1l-one (1.65 g, 0.011 mole; see, forexample, F. Naf et al., Helv. Chim. Acta 1978, 2524) and trimethylsilylchloride (2.8 ml, 0.022 mole) in ether (15 ml) was added dropwise during30 min, at -44°. After an additional 15 min at -40°, the mixture wasallowed to attain 0° during 1 h and then poured into cold aq. NH₄ Cl.Extraction with ether afforded an organic phase which was successivelywashed with H₂ O and sat. aq. NaCl and dried over anhydrous Na₂ SO₄.Concentration under vacuum afforded a raw oil (7 g) consisting of thetrimethylsilyl enol ether of the desired intermediate, which oil wasdissolved in THF (25 ml) and stirred with 10% aq. HCl (1 ml) during 30min. After a further extraction with ether, treatment and purificationby chromatography [SiO₂ (100 g); eluting agent:cyclohexane/ethyl acetate19:1)] afforded(Z)-3-(8'-benzyloxyoct-1'-yl)-2-(2-pentenyl)-cyclopentan-1-one (13:1trans/cis mixture) in the form of a viscous yellow oil (3.1 g; yield30%), which was dried at 50°/1.3 Pa.

NMR (¹ H, 360 MHz): 4.96(t, J=7 Hz, 3H); 1.20-2.40(24H); 7.47(t, J=7 Hz,2H); 4.50(s, 2H); 5.25(m, 1H); 5.42(m, 1H); 7.25-7.35(5H) δ ppm NMR (¹³C): 220.8(s); 138.7(s); 133.4(d); 128.3(d); 127.6(d); 127.5(d);125.5(d); 72.9(t); 70.5(t); 55.1(d); 41.2(d); 38.1(t); 34.7(t); 29.8(t);29.5(2t); 27.1(t); 26.2(t); 25.4(t); 20.6(t); 14.2(q) δ ppm MS: 370 (1,M⁺), 302(10), 279(26), 261(9), 173(10), 151(30), 91(100), 83(25)

Trimethylsilyl iodide was added via a syringe to a stirred solution ofthis pentanone (2.1 g, 5.7 mmole; trans/cis 13:1) in CH₂ Cl₂ (8 ml), atr.t. and under N₂. After 25 min, the mixture was poured into 10% aq.NaHSO₃ (50 ml) and extracted with ether. The organic phase was washedsuccessively with sat. aq. NaHCO₃ and sat. aq. NaCl and dried overanhydrous Na₂ SO₄. Concentration under vacuum afforded the trimethylsilyl ether of the desired intermediate, which was dissolved in THF (15ml) and stirred with aq. 10% HCl during 20 min. Re-extraction withether, treatment and chromatography [SiO₂ (100 g); eluting agent:cyclohexane/ethyl acetate 4:1], followed by bulb-to-bulb distillation,afforded (Z)-3-(8'-hydroxyoct-1'-yl)-2-(2-pentenyl)-1-cyclopentanone(trans/cis 13:1) in the form of a colorless oil (1.25 g; yield 78%).

B.p.: 220°-240° (bath)/5.3 Pa IR (CHCl₃): 3440 (broad), 3015, 2930,2857, 1732, 1462 cm⁻¹ NMR (¹ H, 360 MHz, D₂ O): 4.96(t, J=7 Hz, 3H);1.20-2.40(24H); 3.63(t, J=7 Hz, 2H); 5.25 (m, 1H); 5.42(m, 1H) δ ppm NMR(¹³ C) 221.0(s); 133.5(d); 125.5(d); 62.8(t); 55.1(d); 41.1(d); 38.1(t);34.7(t); 32.7(t); 29.8(t); 29.6(t); 29.4(t); 27.1(2t); 25.8(t); 25.4(t);20.6(t); 14.2(q) δ ppm MS: 280 (2, M⁺), 212(13), 151(36), 124(33),109(15), 95(40), 83(100)

Jones reagent (H₂ CrO₄ /aq. H₂ SO₄ ; 4 ml of a solution 2.5M) was addeddropwise to a stirred solution of the latter cyclopentanone (1.1 g, 3.9mmole; trans/cis 13:1) in acetone (20 ml) at 20°, under N₂. After afurther 40 min at 22°-24°, the mixture was poured into water andextracted with ether. The organic phase was successively washed with H₂O, 10% aq. NaCl and sat. aq. NH₄ Cl, dried over anhydrous Na₂ SO₄ andconcentrated under vacuum. The resulting yellow oil (2.1 g) was purifiedby chromatography [SiO₂ (50 g); eluting agent:cyclohexane/ethyl acetate1:1.5)] to afford the desired(Z)-3-oxo-2-(2-pentenyl)-1-cyclopentaneoctanoic acid (trans/cis 13:1) inthe form of a viscous pale yellow oil (1 g; yield 87%).

IR (CHCl₃): 3300 (broad), 3020, 2931, 1732, 1710 cm⁻¹ NMR (¹ H, 360 MHz,D₂ O): 0.96(t, J=7 Hz, 3H); 1.20-2.40(24H); 5.24(m, 1H); 5.42(m, 1H) δppm NMR (¹³ C): 221.1(s); 179.3(s); 133.5(d); 125.5(d); 55.1(d);41.1(d); 38.1(t); 34.7(t); 33.9(t); 29.6(t); 29.2(t); 29.0(t); 27.1(t);27.0(t); 25.4(t); 24.7(t); 20.6(t); 14.2(q) δ ppm MS: 294 (<0.5, M⁺),226(1), 151(8), 124(16), 95(26), 83(100)

EXAMPLE 4

We proceeded according to general method II described in Example 3,using the microorganisms cited in the following table. In this table,compounds A, B, C and D are the same as cited in Example 3. The resultsindicated relate to the product obtained after 24 h reaction. The molaryields are indicated in % relative to the added starting acid, i.e.(Z)-3-oxo-2-(2-pentenyl)-1-cyclopentaneoctanoic acid (trans/cis 13:1, 20mg, ˜65 μmole).

The results in this table show that in the case of the reactions carriedout with Rhodococcus rhodochorus, Arthrobacter petroleophagus andAspergillus niger, pratically no kinetic resolution between the (+) and(-) enantiomers of the substrate is observed, whereas in all other casesthe amount of (+)-trans,(Z)-3-oxo-2-(2-pentenyl)-1-cyclopentaneaceticacid formed (corresponding to compound A) is larger than that of its(-)-trans enantiomer. This therefore indicates that the correspondingmicroorganisms are capable of kinetic resolution among the substrateenantiomers. Furthermore, it is apparent from this table that theresolution takes place essentially at the stage of the conversion of(Z)-3-oxo-2-(2-pentenyl)-1-cyclopentanebutanoic acid (corresponding tocompound B) into (Z)-3-oxo-2-(2-pentenyl)-1-cyclopentaneacetic acid. Theresults of identical experiments carried out by way of Cylindrocarponcandidum CBS 132,25, Arthrobacter atrocyanus DSN 20127, Streptomycesbacilliaris DMS 40598, Arthrobacter ureafaciens DMS 419 or Streptomycesrosechromogenus seem to indicate that these microorganisms requirereaction times above 24 h to carry out the β-oxidation of the substratescited in the table, under the described reaction conditions.

    TABLE III      -      (-)-         trans-A/      Weight Acid Molar yield (%) A/ (+)-      of the fraction - D C B A Total % Chiral GC (-)-trans-C/ % Chiral GC     (-)-trans-B/ % Chiral GC trans-A % GC       Final extract Molar (trans + (trans + (trans + (trans + acids Trans-D     Cis-D Trans-C (+)-trans-C Cis-C Trans-B (+)-trans-B Cis-B Trans-A Enant. C     is-A      Microorganism pH (mg) yield (%) cis) cis) cis) cis) (%) (+ and -) (+     and -) (+ and -) Enant. ratio (+ and -) (+ and -) Enant. ratio (+ and -)     (+ and -) ratio (+      and -)      Rhodococcus 7.52 28.2 74.01 0.00 0.56 5.60 67.86 91.69      93.1  6.9     93.9 0.97 6.1      rhodochorus      ATCC 4273      Nocardia 8.01 30.0 85.64 0.00 0.78 19.97 64.90 75.77      89.9 8.24     10.1 93.9 0.65 6.1      calcarea      DSM 43188      Rhodococcus 7.83 17.4 83.46 0.71 0.58 26.17 56.02 67.11   100  0 95.7     4.3 93.0 0.41 7.0      erytropolis      DSM 1069      Arthrobacter 7.73 15.7 70.30 3.77 0.93 9.81 55.79 79.35 93.3 6.7     79.0 2.31 21.0 93.8 0.97 6.2      petroleophagus      ATCC 21494      Rhodococcus 7.34 22.5 70.03 0.00 0.52 15.81 53.71 76.68      93.7  6.3     93.1 0.59 6.9      sp. DSM 6344      Aspergillus 5.34 14.2 33.41 0.59 0.37 1.95 30.50 91.29 93.7 6.3 82.0     18.0 100.0 4.80 0 93.9 1.06 6.1      niger      ATCC 9142      Saccharomyces 8.40 36.0 30.97 0.22 1.76 20.15 8.85 28.57   86.5 2.0     13.5 91.4 2.88 8.6 96.4 0.16 3.6      cerivisiae      (Canada)      Saccharomyces 8.22 35.0 42.71 16.90 3.28 18.11 4.42 10.34 91.6 8.4 80.6     5.2 19.4 89.2 2.13 10.8 100.0 0.23 0      cerivisiae      (Suisse)      Mycobacterium 7.54 23.7 81.40 64.06 14.48 2.44 0.43 0.53 92.8 7.2 93.6     0.4 6.4 100.0 0.32 0 100.0 1.18 0      phlei      DSM 750

EXAMPLE 5

We proceeded according to the general method I described in Example 1,by adding 100 mg of 2-oxo-1-cyclopentanehexanoic acid (obtained bysaponification with NaOH of its ethyl ester available from Janssen) to asuspension of Saccharomyces cerevisiae. The acid fraction (38.6 mg) ofthe reaction product was esterified to afford a mixture of esterscontaining 25% by weight of the methyl ester of the starting acid, aswell as the two following compounds:

methyl 2-oxo-1-cyclopentaneacetate (12.9%)

GC-MS: 156 (41) [M⁺ ], 125 (100) [M--OCH₃ ⁺ ], 124 (97), 113 (34), 97(47), 83 (54) [C₅ H₆ O+H⁺ ], 74 (48) [M--CH₂ COOCH₃ ⁺ ], 59 (49)

methyl 2-oxo-1-cyclopentanebutyrate (41.2%)

GC-MS: 184 (13) [M⁺ ], 153 (19) [M--OCH₃ ⁺ ], 152 (53), 137 (18), 124(28), 101 (4) [M--(CH₂)₃ COOCH₃ ⁺ ], 97 (48), 84 (100) [C₅ H₈ O], 74(47),55 (38)

I claim:
 1. Process for the preparation of cyclic ketones of formula##STR10## wherein the dotted line indicates the location of a single ordouble bond, m represents an integer from 0 to 3 and n an integer from 0to 10, each of the symbols R, which can be identical or different,stands for hydrogen or for a saturated or unsaturated, linear orbranched, hydrocarbon radical having 1 to 6 carbon atoms, and each ofthe substituent groups can be located in any available position of thering, which process comprises adding a substrate containing one orseveral cyclic carboxylic derivatives of formula ##STR11## wherein thedotted line and the symbols R and m have the meaning indicated informula (I), p>n+2 and is defined as being an even integer when n iseven and an odd number when n is odd, to a culture of a microorganismcapable of β-oxidising the fatty acid chain of said derivatives, andcontacting said substrate with said culture for an amount of timesufficient to form at least one of said ketones (I) which is thenextracted from the reaction medium.
 2. Process according to claim 1,which comprises adding a substrate containing one or several derivativesof formula ##STR12## wherein the dotted line indicates the location of asingle or double bond and p>n+2 and is odd.
 3. Process according toclaim 1, which comprises adding a substrate containing one or severalderivatives of formula ##STR13## wherein p has the meaning indicated informula (II).
 4. Process according to claim 1, which further comprisesselecting the microorganism to be one from the Saccharomyces orRhodococcus genus.
 5. Process according to claim 1, which furthercomprises selecting the microorganism from the group consisting ofRhodococcus rhodochorus, Rhodococcus erytropolis, Rhodococcus sp.,Nocardia calcarea, Arthrobacter petroleophagus, Arthrobacterartrocyanus, Arthrobacter ureafaciens, Aspergillus niger, Saccharomycescerivisae, Mycobacterium phlei, Streptomyces viridosporus, Streptomycesrosechromogenus, Streptomyces bacilliaris, Cyclindrocarpon candidum,Escherichia coli, Hansenula polymorpha, Pseudomonas Sp., Serratiamarcesens and Aspergillus oryzae.
 6. Process according to claim 5, whichfurther comprises adding (Z)-3-oxo-2-(2-pentenyl)-1-cyclopentaneoctanoicacid to a culture of a microorganism selected from the group consistingof Rhodococcus rhodochorus, Rhodococcus erytropolis, Rhodococcus sp.,Nocardia calcarea and Arthrobacter petroleophagus, to predominantly form(Z)-3-oxo-2-(2-pentenyl)-1-cyclopentaneacetic acid.
 7. Process accordingto claim 5, which further comprises adding3-oxo-2-pentyl-1-cyclopentaneoctanoic acid to a culture of amicroorganism selected from the group consisting of Serratia marcesensand Aspergillus niger, to predominantly form3-oxo-2-pentyl-1-cyclopentaneacetic acid.
 8. Process according to claim1, which further comprises subsequently esterifying the formed productto obtain the corresponding ester.
 9. Process according to claim 1,wherein the substrate is added to the culture of the microorganism in amedium devoid of any other nutritive source.